Although anti-retroviral therapy (ART) effectively controls HIV-1 infection, it has several limitations including uncomfortable side effects, low accessibility, and high cost. The development of a sterilizing or functional cure would address many of these limitations. The primary obstruction to a cure is the presence of persistent, replication-competent HIV in patients on ART. Recently, several clinical trials have shown that they can target persistent HIV with small-molecule inhibitors. However, most of this work has focused on virus that is pharmacologically easily accessible in the circulatory system. The next important step is to specifically determine where reservoirs of persistent HIV exist in patients on ART so that new therapies can be targeted to these locations. This project focuses on the lymph node follicles, which are a known source of persistent HIV-1. However, the role of the individual cell types in the lymph node follicles in the persistence of HIV is not fully defined. The lymph node follicles contain follicular-dendritic cells (FDCs) that can sequester replication competent virus on their cell surface, which could then potentially infect susceptible follicular T helper cels (TFHs). Depending on whether the persistent HIV in the lymph nodes is primarily seeded by latent HIV proviruses in target cells or whether FDC-associated virions are contributing to persistence drastically affects how future strategies aimed at clearing this virus should be developed. To address this question, we have collected lymph node follicles from study participants on long-term ART and will characterize the morphology of the FDCs and TFHs using microscopy. Then, we will isolate both the FDC and the TFHs using laser-capture microdissection and will use single-genome sequencing and single-proviral sequencing to characterize the virus in these cells and determine whether HIV sequestered on FDCs acts as a reservoir of infectious virus in patients on ART and whether this virus can then disseminate to other known reservoirs of persistent HIV. By defining the role of FDCs in the persistence and dissemination of HIV in patients on long-term ART, this important work will contribute to the development of future HIV eradication clinical trials and allow them to focus and direct therapeutics to the anatomical locations that are the most relevant sources of persistent HIV. Additionally, this project will specifically address the NIAIDs area of emphasis to Cure HIV Infection priority one to determine if additional reservoirs of HIV infection exist.